When skin damage such as burn occurs, one should be most careful about bacterial infection from the skin injured by burn and the like. The dead skin is particularly susceptible to extensive proliferation of miscellaneous germs. Therefore, the dead skin must be removed to ensure that no miscellaneous germs will proliferate. However, removal of the skin provides a site for bacterial infection. In order to prevent such bacterial infection, the site from which the skin has been removed must be masked with a suitable material to avoid the ingress of bacteria. Masking materials that can be used for this purpose include synthetic polymer materials and a cultured skin. However, synthetic polymers can potentially cause rejection and the like and are not preferred as skin grafts. On the other hand, the cultured skin is a portion of the normal skin of the patient himself that has been cultured to a desired size, so it can be used without any possible inconvenience such as rejection and may well be described as the most natural masking material.
Cell culture of the kind described above has conventionally been performed on the glass surface or the surfaces of synthetic polymers subjected to various treatments. For example, a variety of vessels and the like that are made of polystyrene and which have been subjected to surface treatments such as irradiation with γ-rays and silicone coating are extensively used as cell culture vessels. Cells that have been cultured and grown in such cell culture vessels are peeled off and recovered from the surface of the vessel by treatment with proteolytic enzymes such as trypsin or chemicals.
However, several defects have been pointed out to exist in the recovery of grown cells by such treatment with chemicals; for one thing, the process of treatment is complicated enough to increase the possibility of contamination by impurities; second, the grown cells are denatured or damaged by the chemical treatment and their inherent functions are injured in some cases. With a view to overcoming these difficulties, several techniques have so far been proposed.
Japanese Patent Publication No. 23191-1990 describes a method of producing transplantable membranes of keratinous tissue by culturing keratinocytes from a human newborn in a culture vessel under conditions where a membrane of keratinous tissue is formed upon the surface of the vessel and enzymatically peeling off the membrane of keratinous tissue. Disclosed specifically is a technique in which 3T3 cells are grown as a feeder layer and piled up in multiple layers and the resulting cellular sheet is recovered with a proteolytic enzyme Dispase. However, the method described in the patent has had the following defects.    (1) Dispase is of bacterial origin and the recovered cellular sheet must be washed thoroughly.    (2) The conditions of treatment with Dispase differ from one cultured cell to another and the treatment requires great skill.    (3) The cultured epidermal cells are activated pathologically by Dispase treatment.    (4) Dispase treatment decomposes the extracellular matrix.    (5) As a result, the diseased part to which the recovered cellular sheet has been grafted is susceptible to infection.
Japanese Patent Laid-Open No. 192138/1993 describes a method of culturing skin cells by preparing a cell culture support having a substrate surface coated with a polymer whose upper or lower critical solution temperature in water is 0˜80° C., culturing skin cells on the cell culture support at a temperature either below the upper critical solution temperature or above the lower critical solution temperature and thereafter peeling off the cultured skin cells by bringing the temperature to either above the upper critical solution temperature or below the lower critical solution temperature. In this method, temperature change is employed to peel off the cells from the culture substrate coated with the temperature-responsive polymer; however, the method does not permit efficient cell peeling and the obtained cellular sheet has had a lot of structural defects.